DESCRIPTION (Taken from the Application): This proposal aims to develop, integrate, and test all of the constituents for carrying out cost-effective, high-throughput structural genomics research for both prokaryotic and eukaryotic systems. The objectives are two-fold: (1) To develop and test experimental and computational strategies for carrying out a cost-effective, high-throughput structural determination of proteins by X-ray crystallography and NMR methods. (2) To apply these strategies to scan the entire genome of an organism at a rapid pace. The eukaryotic organism, Caenorabditis elegans, and an ancestrally-related prokaryotic microorganism having a small genome, Pyrococcus furiosus, are selected as representative genomes. As human cDNAs from the NCl sponsored genome project become available, they will be included in the study with the ultimate goal being structural characterization of all proteins of the human genome. By selecting both prokaryotic and eukaryotic genomes we should be able to explore the full breadth of obstacles to high-throughput structure determination of gene products from any genome. The Pilot Center has three working groups: Protein Production Group, NMR Group, and X-ray Crystallography Group. Using a robotic approach, the protein production group plans to supply approximately 80 proteins/week (1-5 mg each) for both NMR and X-ray crystallographic studies. While the NMR Group has developed methods that potentially could lead to high-throughput methods, the X-ray crystallography group has developed innovative approaches in three areas of protein crystallography. They are: crystallization of proteins using microgram quantity of sample, increased data collection efficiency 3 to 5 times at a synchrotron site, and a breakthrough in phase determination using sulfur anomalous scattering signal. The new process of crystallization will significantly impact the structural genomics in that essentially all proteins, even those that can be expressed in microgram quantities will be candidates for crystallographic studies. The latter is significant in terms of cost-effectiveness for large-scale structure determination as it could allow one to solve a protein crystal structure routinely without the need of substituting the sulfur by selenium atom, saving both time and cost. This proposal aims to integrate these developments and others to produce a system for cost-effective, high-throughput structure determination on both prokaryotic and eukaryotic systems. Once fully developed, this system will dramatically change our speed of structure determination. It will have broad implications to medical science and biotechnology development in this nation.